The present invention relates to luminescence indicators for determining cations, in particular alkali ions, of liquid, in particular aqueous, media. In effecting such determination, the substance to be determined (=analyte) is contacted with a luminescence indicator (=luminophore-ionophore) having a luminophoric moiety and an ionophoric moiety, which ionophoric moiety reacts with the analyte present in the sample, wherein the luminophoric moiety changes its luminescence properties, after which the luminescence is measured and the concentration and activity of the analyte are deduced, i.e. the cation is determined, utilizing the test reading.
A determination method of this type is based on the so-called "PET effect". This latter term denotes the transfer, induced by photons, of electrons (photoinduced electron transfer=PET) from the ionophoric moiety or ionophore, respectively, to the luminophoric moiety or luminophore, respectively, which leads to a decrease in the (relative) luminescence intensity and the luminescence decay time of the luminophore. Absorption and emission wavelengths, however, remain basically unaffected in the process (J. R. Lakowicz in "Topics in Fluorescence Spectroscopy", Volume 4: Probe Design and Chemical Sensing; Plenum Press, New York & London (1994)).
By the binding of ions to the ionophore, the PET effect is partly or completely inhibited, sb that there is an increase in the luminescence of the luminophoric moiety. Hence, the concentration or the activity of the ion to be determined can be deduced by measuring the change in luminescence properties, i.e. luminescence intensity and/or luminescence decay time.
From U.S. Pat. No. 5,516,911, fluorescence indicators for determining intracellular calcium are known which carry fluorescent substituents capable of acting as optical indicators.
A determination method is also known from U.S. Pat. No. 5,439,828, where diaza-cryptands are utilized as the luminophore-ionophore, which diaza-cryptands have been functionalized as fluorophores with fluorescent coumarins and, depending on their structure, are selective for lithium, sodium or potassium ions. It is stated that these luminophore-ionophores can be used in sample media of neutral pH and are even the preferred choice in such systems.
Yet, research (Frank Kastenholz, Inaugural Dissertation, University of Cologne, 1993, FIG. 32, p. 54) has shown that in the physiological pH range the fluorescence signal depends significantly on the pH of the sample and increases considerably with decreasing pH, even from pH 7.4 onwards. This affects the accuracy of a determination carried out in a biological sample. Moreover, the compounds that are being used have the disadvantage that the employed coumarins show absorption wavelengths of about 336 nm and hence cannot be excited by commercial LEDs.
These disadvantages also apply to the luminophore-ionophores mentioned in U.S. Pat. No. 5,162,525.
From Tetrahedron Letters, Volume 31, No. 36, pp. 5193-5196 (1990), diaza-cryptands are known in which the two nitrogen atoms are bound to a respective aromatic ring each, i.e. are aryl nitrogens and aniline-type nitrogens, respectively. Research conducted by the applicant has shown that these diaza-cryptands are not suited for determining potassium ions if they are present in the physiological range of concentration and at physiological pH values of the blood (7.0-7.6).
U.S. Pat. No. 4,774,339, U.S. Pat. No. 5,187,288, U.S. Pat. No. 5,274,113 and U.S. Pat. No. 5,248,782 describe fluorescent dyes containing dipyrrometheneboron difluoride as the parent substance and its derivatives with reactive substituents for covalent binding to biomolecules.
From U.S. Pat. No. 5,433,896, fluorescent dyes are known which contain 1-[isoindolyl]methylene-isoindiol as the parent substance.
Dye-conjugates of dipyrrometheneboron difluoride, wherein at least one of the reactive substituents covalently binds a dye molecule to a specific binding pair member, f.i. a nucleotide or a protein, are described in U.S. Pat. No. 5,451,663.